A secreted effector with a dual role as a toxin and as a transcriptional factor

Bacteria have evolved multiple secretion systems for delivering effector proteins into the cytosol of neighboring cells, but the roles of many of these effectors remain unknown. Here, we show that Yersinia pseudotuberculosis secretes an effector, CccR, that can act both as a toxin and as a transcriptional factor. The effector is secreted by a type VI secretion system (T6SS) and can enter nearby cells of the same species and other species (such as Escherichia coli) via cell-cell contact and in a contact-independent manner. CccR contains an N-terminal FIC domain and a C-terminal DNA-binding domain. In Y. pseudotuberculosis cells, CccR inhibits its own expression by binding through its DNA-binding domain to the cccR promoter, and affects the expression of other genes through unclear mechanisms. In E. coli cells, the FIC domain of CccR AMPylates the cell division protein FtsZ, inducing cell filamentation and growth arrest. Thus, our results indicate that CccR has a dual role, modulating gene expression in neighboring cells of the same species, and inhibiting the growth of competitors.


Reviewer #1 (Remarks to the Author):
In the study "Cell-to-cell communication mediated by a T6SS secreted transcriptional regulator" ", the authors use a multidisciplinary approach, including genetic, biochemical and biochemical assays, to functionally and structurally characterize the CccR toxin of Yersinia pseudotuberculosis (Yptb). CccR is a FIC protein that additionally presents an HTH domain, so it can act as a transcription factor. In the manuscript the authors show that the protein is actually bifunctional, since once excreted into the medium through the T6SS system, it can act as a toxin in competing bacteria by the AMPylation of the cell division protein FtsZ. To mediate this function CccR is imported in the target bacteria engaged to FhuA. Meanwhile, CccR functions as a signaling protein in kin bacteria acting as a transcriptional factor. The work is multidisciplinary and very well executed, the results are clear, the manuscript is well written and the logic is easy to follow. Taken together, the novelty and originality of the results presented as well the large number and high technical quality of experimental approaches used to confirm these results, the manuscript shows the sufficient entity to be published in Nature Communications. However, in my view, some of the (main) conclusions of the manuscripts are not completely supported and/or checked and, therefore, the manuscript needs additional data addressing the following concerns before to be accepted for the publication.
Once of the major contributions of the study is to demonstrate the bifunctional character of CccR, acting as both a toxin and a transcriptional regulator in receptor cells. With these results, the authors propose a model for the dual role of CccR in bacterial competition and cell-to-cell communication ( fig 6) where the toxin activity is working in nonself recipient cells (competitors) mainwhile the signaling activity (transcriptional regulator) acts in kin cells. However, the selectivity of this dual activity is supported by the fact that the authors have not tested both functions in both types of cells (competitor and kin). In other words, could CccR act as a toxin in kin cells? or, oppositely, could CccR act as a transcriptional regulator in competitor cells?
The authors have basically all the tools to confirm both points and thus support the proposed model.
1. Toxin activity in Yptb, it is easy to evaluate whether overexpression of CccR induces filamentation of this bacterium or whether CccR mediates in vitro the AMPylation of Yptb FstZ. In addition, the authors know the AMPylation site for E. coli FstZ (Thr8), so they could evaluate in silico how conserved is this site in other strains and then discuss about possible range of target strains.
2. Regulatory (signaling) activity in competitor cells. The authors make a great effort throughout the manuscript to map the DNA binding site of CccR, as well as to unveil the genes regulated by this protein in Yptb. However, with all this information available they do not evaluate whether CccR can regulate genes in competing strains once it is imported. If the authors know the DNA binding site, they could locate these sequences in the E. coli genome and design assays with reporters, such as those used in the study, to evaluate whether regulation also exists for E. coli genes. In addition, and related to the regulatory capacity of CccR, the authors do not make an intensive analysis of the RNAseq data. Basically, it is a description of the experimental results indicating the number of up-and down-regulated genes and the pathways in which these genes are involved. It is quite possible that a more detailed analysis will be presented in future papers, but the authors should discuss the data in more depth here. At least the authors should localize the CccR binding boxes in the regulated genes to assess whether it is a direct or indirect regulation, even more so when CccR seems to act as a transcriptional repressor and the RNAseq results show a large number of upregulated genes by CccR.
Also related with the regulatory activity of CccR, the authors show from the structural data the weak dimerization surface of the protein, which is completely abolished with mutations at only two positions (N13 and K175). The authors could analyze whether this mutant is defective in regulatory capacity (CccR should be dimer to recognize the palindromic binding site), but not in toxin activity. This data would reinforce the independence of both activities and may imply additional levels of regulation for the system.
Finally, and related with the previous point, I consider that the authors make little use of the structural data (which must have required a major effort), other than to explain structurally the infrequent DNA-binding site (two inverted repeats of the 10-bp separated by a spacer of 26 bp) of CccR . The authors could also structurally confirm the catalytic activity of this protein by docking the substances and justifying why the H192A mutation (widely used in the studies) is catalytically inactive.
Reviewer #2 (Remarks to the Author): In this exciting study, the authors made an unusual discovery of the function of a T6SS in Yersinia pseudotuberculosis: it secrets the multifunctional protein CccR into the extracellular milieu and the protein is take up by competitive or kin cells via binding to the transporter FhuA. Most interestingly, whereas CCCR functions as an AMPylator to inhibit cell division by attacking FtsZ, it acts as a transcriptional regulator to modulate the expression of large set of genes, making the bacterium better fit in hostile environments. Overall, this is an excellent study, the experiments are well-designed, and the results are of high quality. The findings have significantly expanded our appreciation of the function of T6SSs and their substrates. I read with great interest the manuscript entitled "Cell-to-cell communication mediated by a T6SS secreted transcriptional regulator". The authors of this study demonstrate that CccR, a T6SS effector from Yersinia pseudotuberculosis, acts as a contactdependent and contact-independent toxin against prey cells as well as exerts transcriptional control on sister cells in the producing population. Overall, this is a solid study presenting novel and exciting results that make a significant contribution to the T6SS field.
The majority of the paper is dedicated on the action of CccR on prey cells. The experiments performed in this part of the study are excellent and beautifully presented. The authors back each of their claims with multiple experiments using diverse approaches and complementary techniques that are well-designed and executed. For this part of the work, I only have a few minor suggestions for improvement: -It would be useful to have some quantifications for the data presented in figures 1d, 2e and extended data figures 1a and 1f. It is clear that the cells are elongated and filamented upon exposure to CccR, nonetheless a graph roporting on cell length would make it more compelling.
-Line 119: The phrase "These results demonstrated that CccR is a FIC toxin." is rather absolute. I would re-phrase to "These results show that CccR exerts FIC toxicity when expressed in E. coli".
-Why did the authors choose to use DH5 alpha cells in their colonization assays. This is a strange choice of strain for in vivo experiments so some justification would be useful.
-Line 183: The fact that the N-terminal portion of CccR is important for internalization into prey cells further support that this toxin behaves in a colicin-like manner when it comes to prey cell entry. This could be indicated in the text.
A smaller part of the manuscript is dedicated on the action of CccR on the producing population and its sisters and shows proof that this effector acts as a negative autoregulator. In my view, the data presented on the activity of the effector on kin cells is robust. That said, this section left me with many unanswered questions, some of which could be investigated by the authors, especially considering that this is the main finding displayed in the title of the manuscript. Please find my questions and suggestions below: -It is not discussed whether CccR has an immunity protein. Indeed, it must not since it can act on the producer and its sisters. In this case, how does the producer counteract the action of this toxin on its own FtsZ? Does CccR not interact with FtsZ from Yersinia pseudotuberculosis? Or does CccR not have any activity against that FtsZ analogue? The authors are experts in all of the techniques required to investigate whether CccR acts on Yersinia pseudotuberculosis FtsZ. In addition, in the case where this toxin does not act on the target of its producer, I think itwould strengthen the manuscript to show that it does act of FtsZ analogues of other prey bacteria beyond E. coli.
-When CccR acts on the producer does that happen while it is produced inside the cell, or does it get re-internalized after secretion through the T6SS? It is somewhat shown that it is the latter using the reporter strain. That said, identifying the receptor in Yersinia and doing similar experiments as the Transwell assays presented currently in the manuscript with a mutant strain for that receptor would give more definitive answers, since both scenarios could be at play. Once more, the authors have the expertise to investigate this.
-I can see that this toxin affects the producer in a very specific manner and the authors discuss this in the context of fitness of the producer cell (Fig. 6). However, the autoregulatory function of CccR in the absence of an immunity protein could also be protective of the producer and its sisters. The authors do not touch on what part of the T6SS apparatus delivers this toxin. But one can imagine a scenario where while it is produced, CccR binds to a T6SS component (Hcp or VgrG) and that prevents it from being toxic. So, its repressor function could just aim to prevent toxicity in cases where its binding to T6SS components is not sufficient for some reason. The same can be said for sister cells; in the presence of many prey cells a lot of this toxin will be internalized to kill them. In the absence of prey, it could be acting as a signaling molecule to inform the population that its production is not necessary since it is mainly sisters that internalize it. Those hypotheses do not negate that it is acting as a signaling molecule in addition to being a toxin. I just feel that the discussion is particularly brief and is using a very specific angle when it comes to cell-to-cell communication; it should be re-worded to be more inclusive of other possibilities that could still belong to the realm of cell-tocell communication. Finally, if the authors wish to claim that the repressor function of CccR increases fitness of the producer they could try and show this using their colonization model and producers with wild-type regulation, as well as disrupted regulation (for example using their palindrome sequences) and assess how well these two strains perform against E. coli in vivo.
Overall, I would like to re-iterate that this is a very nice study and with some changes it could made a big contribution to the T6SS field. Thank you for the opportunity to review this very nice paper! Reviewer #4 (Remarks to the Author): This manuscript focuses on a Yersinia pseudotuberculosis T6SS secreted effector, CccR, containing two domains: a FIC domain and a HTH domain. In a systematic manner the authors demonstrated that this effector functions as a FIC toxin which mediates growth inhibition, it is secreted via T6SS-3 and 4 in a contact-dependent and contactindependent manner. In relation to its contact-independent effect, it was found that FhuA mediates CccR entry into target cells. The main target of CccR in E. coli was found to be FtsZ. CccR was found to bind and AMPylate FtsZ, thus leading to inhibition of the FtsZ GTPase activity and its polymerization. Additionally, the crystal structure of CccR was determined, it was found to autoregulate its own expression using an invertedrepeat sequence located in its regulatory region. Moreover, CccR was shown to regulate gene expression also after it was delivered into neighboring kin cells. Thus, the authors demonstrated that CccR functions on competing cells (growth inhibition using its FIC domain) as well as on kin cells (transcriptional regulation using its HTH domain).
Major comments: 1. Fig. 3D and Fig. 3E, in both panels the effect of CccR on FtsZ was examined. In Fig. 3D the CccR effect on FtsZ GTPase activity and in Fig. 3E the CccR effect on FtsZ polymerization. In both assays, the concentration of CccR used was very high, and this does not fit the function of CccR as a protein harboring enzymatic activity -AMPylation. In the GTPase activity assay in order to reduce the FtsZ activity a ratio of 1:1 between the substrate (FtsZ) and enzyme (CccR) was required. The requirement for such a ratio is expected in the case where the binding of CccR to FtsZ per se results in the reduction of FtsZ GTPase activity but not if CccR enzymatic activity makes the effect. In the FtsZ polymerization assay, there was a need for 100microM of CccR in order to inhibit the polymerization of 10microM of FtsZ, this clearly does not fit the hypothesis that AMPylation of FtsZ results in the inhibition of polymerization, but it rather seems that the binding between the two proteins led to the observed inhibition. This issue should be resolved for example by using low concentration of CccR over a time course to examine its effect on FtsZ, or to use clean AMPylated-FtsZ in the two assays (without CccR).
2. Fig. 5C (lines 339-344) -The authors generated an E. coli strain with the cccR gene containing a mutation in the palindromes of the regulatory element (PcccRm) which drastically increased CccR expression. The protein band in Fig. 5C in the relevant lane is very strong. How such a strain can be alive? The strain produces high level of a potent toxin which AMPylates FtsZ, how can this strain replicate? this result does come together with the other information presented in the manuscript regarding CccR effect on E. coli.
3. The authors identified the regulatory element recognized by CccR, which is rather special (line 329). The RNA-seq analysis resulted in the identification of 447 genes which were up-or down regulated after CccR delivery. It is highly likely that very few (if any) of these genes harbor the CccR regulatory element, therefore it is not clear how CccR affects their expression. It might be that CccR function as a FIC toxin is responsible for the effect (induction of stress) and not its function as a transcriptional regulator. The RNA-seq analysis should be repeated with a CccR mutated in the FIC domain to validate that the change in gene expression occurs due to CccR function as a transcriptional regulator. 4. It was not described in the manuscript if there is a known antitoxin for CccR. The FtsZ proteins of E. coli and Yersinia pseudotuberculosis are identical at the N-termini where CccR AMPylates FtsZ. Therefore, it is expected that Yersinia pseudotuberculosis will harbor an anti-toxin which inhibit CccR activity. Is there any information about the CccR antitoxin?

Reviewer #1 (Remarks to the Author):
In the study "Cell-to-cell communication mediated by a T6SS secreted transcriptional regulator" ", the authors use a multidisciplinary approach, including genetic, biochemical and biochemical assays, to functionally and structurally characterize the CccR toxin of Yersinia pseudotuberculosis (Yptb). CccR is a FIC protein that additionally presents an HTH domain, so it can act as a transcription factor. In the manuscript the authors show that the protein is actually bifunctional, since once excreted into the medium through the T6SS system, it can act as a toxin in competing bacteria by the AMPylation of the cell division protein FtsZ. To mediate this function CccR is imported in the target bacteria engaged to FhuA. Meanwhile, CccR functions as a signaling protein in kin bacteria acting as a transcriptional factor. The work is multidisciplinary and very well executed, the results are clear, the manuscript is well written and the logic is easy to follow.
Taken together, the novelty and originality of the results presented as well the large number and high technical quality of experimental approaches used to confirm these results, the manuscript shows the sufficient entity to be published in Nature Communications. However, in my view, some of the (main) conclusions of the manuscripts are not completely supported and/or checked and, therefore, the manuscript needs additional data addressing the following concerns before to be accepted for the publication.
Once of the major contributions of the study is to demonstrate the bifunctional character of CccR, acting as both a toxin and a transcriptional regulator in receptor cells. With these results, the authors propose a model for the dual role of CccR in bacterial competition and cell-to-cell communication (fig 6) where the toxin activity is working in nonself recipient cells (competitors) mainwhile the signaling activity (transcriptional regulator) acts in kin cells.
However, the selectivity of this dual activity is supported by the fact that the authors have not tested both functions in both types of cells (competitor and kin). In other words, could CccR act as a toxin in kin cells? or, oppositely, could CccR act as a transcriptional regulator in competitor cells? The authors have basically all the tools to confirm both points and thus support the proposed model.

Response:
Thank you for your positive feedback and insightful comments regarding our manuscript. As required, we have provided additional experimental data in the revised manuscript to address the concerns you raised.
1. Toxin activity in Yptb, it is easy to evaluate whether overexpression of CccR induces filamentation of this bacterium or whether CccR mediates in vitro the AMPylation of Yptb FstZ. In addition, the authors know the AMPylation site for E. coli FstZ (Thr8), so they could evaluate in silico how conserved is this site in other strains and then discuss about possible range of target strains.

Response:
As suggested, we have tested the toxin activity of CccR in kin cells (Y. pseudotuberculosis) and the results showed that expressing of CccR did not result in growth arrest and cell filamentation of this bacterium (Respond Fig. 1a-b, new Supplementary Fig.   2a-b), implicating the existence of an unknown immunity mechanism to protect the CccR producing kin cell from intoxication.
Sequence alignment analysis showed that the Thr 8 residue is conserved in bacteria of Enterobacteriaceae but not in other families (Respond Fig. 1c, new Supplementary Fig. 6d).
We have added these results to the main text and discussed about possible range of target strains in the discussion section as follow: "It will be interesting to determine whether this FtsZ-targeting mechanism is conserved among Enterobacteriaceae species that contain the conserved Thr 8 residue (Lines 252-254)".
2. Regulatory (signaling) activity in competitor cells. The authors make a great effort Respond Fig. 1. a- Response: Thank you very much for your professional comments. In this study we demonstrated that CccR acts as a transcriptional regulator to auto-repress its own expression not only in its producing cells but also in neighboring kin cells via specific DNA binding, which support the notion that CccR plays a role in cell-to-cell communication.
However, although we have identified 447 genes which were up-or down regulated more than 1.2-fold after CccR delivery by transcriptomics analysis, and the transcriptomics results have been validated by qRT-PCR analysis, we cannot identify putative CccR binding elements in the promoter regions of these regulated genes. So, it is not clear how CccR affects their expression. It might either be that CccR un-specifically recognize these promoters as a transcriptional regulator, or be that CccR function as a FIC toxin is responsible for the effect (induction of stress) for part of these genes. It seems impossible to reveal the global regulatory mechanism of CccR on these genes in this manuscript, but we will work on this topic continually in the future.
Similarly, we cannot identify putative CccR binding elements from E. coli genome. Even we performed a transcriptomics analysis of delivered CccR on E. coli cells, we didn't get valuable information. Given that delivered CccR will inhibit growth of the prey cells, and there is no CccR binding elements can be identified in the E. coli genome, the transcriptional regulator activity of CccR in the prey cells might can be ignored.
We have discussed these issues in the discussion section as follow: "Previously, it was reported that a contact-dependent growth inhibition (CDI) system in Burkholderia thailandensis delivers BcpA, a DNase toxin, to induce changes in the differential expression of 841 genes in immune target cells. Although the molecular mechanism underlying this process, termed contact-dependent signaling (CDS), requires the catalytic activity of BcpA, the details remain unclear. In this study, we provide direct evidence of a role of CccR as a transcriptional regulator that regulates gene expression by directly binding DNA. RNA-seq analysis resulted in the identification of 447 genes that were up-or downregulated more than 1.2-fold after CccR delivery. However, because we cannot identify putative CccR binding elements in the promoter regions of these regulated genes, it is not clear how CccR globally affects their expression. CccR may nonspecifically recognize promoters as a transcriptional regulator, or may function as an FIC toxin that is responsible for the effect (induction of stress). It will be interesting to determine whether CccR can act as a transcriptional regulator in prey cells. However, given that toxic CccR will inhibit prey cell growth, and no CccR binding elements have been identified in the E. coli genome, transcriptional regulator activity of CccR in prey cells might can be ignored like BcpA (Lines 477-493)." 3. In addition, and related to the regulatory capacity of CccR, the authors do not make an intensive analysis of the RNAseq data. Basically, it is a description of the experimental results indicating the number of up-and down-regulated genes and the pathways in which these genes are involved. It is quite possible that a more detailed analysis will be presented in future papers, but the authors should discuss the data in more depth here. At least the authors should localize the CccR binding boxes in the regulated genes to assess whether it is a direct or indirect regulation, even more so when CccR seems to act as a transcriptional repressor and the RNAseq results show a large number of up-regulated genes by CccR.

Response:
We thank the reviewer for this very insightful point. Although we have convincingly demonstrated that CccR acts as a transcriptional regulator to auto-repress its own expression not only in its producing cells but also in neighboring cells via direct DNA binding, we totally agree with you that the analysis of the transcriptomics results is not in-depth. The main problem is that we cannot identify putative CccR binding elements in the promoter regions of these regulated genes. So, it is not clear how CccR affects their expression. It might either be that CccR un-specifically recognize these promoters as a transcriptional regulator, or be that CccR function as a FIC toxin is responsible for the effect (induction of stress) for part of these genes.
It seems impossible to reveal the global regulatory mechanism of CccR on these genes in this manuscript, but we will work on this topic continually in the future. Possibly we need to perform a ChIP-seq to identify genes direct regulated by CccR. So, in this manuscript we mainly focused on the specifically regulation of CccR on its own expression, and these results are sufficient to support the role of CccR in cell-to-cell communication. We toned down the globally regulation part of CccR in the main text and the updated model (Respond Fig. 2, new Fig 6). As suggested, we have also discussed the CccR mediated regulation in-depth in the discussion section (Lines 477-493).

Respond Fig. 2. A model for the dual role of CccR in bacterial competition and cell-to-cell communication.
In CccR-producing donor cells, CccR negatively autorepresses its own expression with a feedback mechanism. Under competition conditions, CccR is released into the extracellular milieu to relieve the autorepression. Released CccR enters target cells by engaging the TonB-dependent outer membrane transporter FhuA in a bacteriocin-like manner. In the presence of many prey cells (Left), secreted CccR is preferentially internalized by prey cells to AMPylate the cell division protein FtsZ, leading to cell filamentation and growth arrest. Following the decrease in prey cells (Right), secreted CccR is internalized by sister cells to repress cccR expression by acting as a transcriptional regulator, informing the population that its production is unnecessary. Under this condition, CccR may also act as a global regulator that regulates expression of genes involved in iron acquisition, motility, and energy production to coordinate bacterial behaviors and increase bacterial fitness. Given that toxic CccR will inhibit the growth of prey cells, the transcriptional regulator activity of CccR in prey cells can be ignored.
4. Also related with the regulatory activity of CccR, the authors show from the structural data the weak dimerization surface of the protein, which is completely abolished with mutations at only two positions (N13 and K175). The authors could analyze whether this mutant is defective in regulatory capacity (CccR should be dimer to recognize the palindromic binding site), but not in toxin activity. This data would reinforce the independence of both activities and may imply additional levels of regulation for the system.

Response:
Thank you for your professional comments. To analyze whether the CccR N13A/K175A mutant is defective in regulatory capacity, we evaluated its effect on repressing the activity of the PcccR promoter. As shown in Respond Fig. 3a (new Fig. 5a The crystal structure of CccR shows S59 and E63 form a stable hydrogen bond with R203 to prevent binding of the ATP substrate for modification (Respond Fig. 4b, Supplementary Fig. 7e) (Lines 332-351)".

Reviewer #2 (Remarks to the Author):
In this exciting study, the authors made an unusual discovery of the function of a T6SS in Response: We thank the reviewer for these valuable suggestions which greatly improved our manuscript. We have re-written the discussion section and proposed an updated model acting as a transcriptional regulator, informing the population that its production is unnecessary. Under this condition, CccR may also act as a global regulator that regulates expression of genes involved in iron acquisition, motility, and energy production to coordinate bacterial behaviors and increase bacterial fitness. Given that toxic CccR will inhibit the growth of prey cells, the transcriptional regulator activity of CccR in prey cells can be ignored".

Reviewer #3 (Remarks to the Author):
I read with great interest the manuscript entitled "Cell-to-cell communication mediated by a T6SS secreted transcriptional regulator". The authors of this study demonstrate that CccR, a T6SS effector from Yersinia pseudotuberculosis, acts as a contact-dependent and contact-independent toxin against prey cells as well as exerts transcriptional control on sister cells in the producing population. Overall, this is a solid study presenting novel and exciting results that make a significant contribution to the T6SS field.
The majority of the paper is dedicated on the action of CccR on prey cells. The experiments performed in this part of the study are excellent and beautifully presented. The authors back each of their claims with multiple experiments using diverse approaches and complementary techniques that are well-designed and executed. For this part of the work, I only have a few minor suggestions for improvement:

Response:
We would like to thank the reviewer for the very positive comments on our study.
We have revised our manuscript in accordance with the reviewer's comments.
-It would be useful to have some quantifications for the data presented in figures 1d, 2e and extended data figures 1a and 1f. It is clear that the cells are elongated and filamented upon exposure to CccR, nonetheless a graph roporting on cell length would make it more compelling. Fig.   1a and 5d, respectively. The quantitation for extended data Figures 1a and 1f was provided in new Supplementary Fig. 1a and 3d, respectively.

Response: The quantitation for Figures 1d and 2e was provided in new Supplementary
-Line 119: The phrase "These results demonstrated that CccR is a FIC toxin." is rather absolute. I would re-phrase to "These results show that CccR exerts FIC toxicity when expressed in E. coli".
Response: Changed as suggested.
-Why did the authors choose to use DH5 alpha cells in their colonization assays. This is a strange choice of strain for in vivo experiments so some justification would be useful.
Response: Thank you very much for your professional comments. We agree with you that E. coli MG1655 was usually used for colonization assays. However, the MG1655 derived -Line 183: The fact that the N-terminal portion of CccR is important for internalization into prey cells further support that this toxin behaves in a colicin-like manner when it comes to prey cell entry. This could be indicated in the text.

Response: Indicated in the text as suggested (Lines 201-203).
A smaller part of the manuscript is dedicated on the action of CccR on the producing population and its sisters and shows proof that this effector acts as a negative autoregulator.
In my view, the data presented on the activity of the effector on kin cells is robust. That said, this section left me with many unanswered questions, some of which could be investigated by the authors, especially considering that this is the main finding displayed in the title of the

Response:
We thank the reviewer for raising this very important issue. As you predicted, we found that CccR can not only interact with Yptb FtsZ (Respond Fig. 5a However, the immunity mechanism of CccR seems very complex and the involved immunity protein needs to be investigated in the future. We have discussed this important Thus, CccR can act as a transcriptional regulator not only in its producing cells, but also in neighboring kin cells once re-internalized after secretion through the T6SS, confirming the prediction that both scenarios could be at play. -I can see that this toxin affects the producer in a very specific manner and the authors discuss this in the context of fitness of the producer cell (Fig. 6). However, the autoregulatory function of CccR in the absence of an immunity protein could also be protective of the producer and its sisters. The authors do not touch on what part of the T6SS apparatus delivers this toxin. But one can imagine a scenario where while it is produced, CccR binds to a T6SS component (Hcp or VgrG) and that prevents it from being toxic. So, its repressor function could just aim to prevent toxicity in cases where its binding to T6SS components is not sufficient for some reason. The same can be said for sister cells; in the presence of many prey cells a lot of this toxin will be internalized to kill them. In the absence of prey, it could be acting as a signaling molecule to inform the population that its production is not necessary since it is mainly sisters that internalize it. Those hypotheses do not negate that it is acting as a signaling molecule in addition to being a toxin. I just feel that the discussion is particularly brief and is using a very specific angle when it comes to cell-to-cell communication; it should be re-worded to be more inclusive of other possibilities that could still belong to the realm of cell-to-cell communication. Finally, if the authors wish to claim that the repressor function of CccR increases fitness of the producer they could try and show this t-test, and differences were considered significant at P < 0.05. ***, P < 0.001. using their colonization model and producers with wild-type regulation, as well as disrupted regulation (for example using their palindrome sequences) and assess how well these two strains perform against E. coli in vivo.
Response: Thank you very much for these excellent suggestions which greatly improved our manuscript! We have provided some new results and re-worded the discussion section to be more inclusive of other possibilities as follow: "However, one can imagine that when CccR is produced, it may be bound by T6SS components (Hcp, PAAR or unknown chaperones) to prevent it from being toxic. Its repressor function could aim to prevent toxicity in cases where its binding to T6SS components is insufficient. The same is true of sister cells. In the presence of many prey cells, secreted CccR proteins will be preferentially internalized by prey cells to kill them. After the prey cells are killed, secreted CccR protein can serve as a signal molecule to inform the population that its production is unnecessary, because it is mainly internalized by sister cells in these conditions. This hypothesis is partially supported by the findings that CccR exhibited significant higher affinity to E. coli c, Comparison of the interaction of CccR with FhuA from E. coli and Yptb with bacterial two-hybrid assay.
Despite repeated attempts, we were unable to construct a regulation disrupted mutant using palindrome sequences (possibly because the auto-repress mechanism is crucial for immunity), which precluded us to assess the repressor function of CccR in increasing the fitness of the producer. So, we toned down this part in the main text and in the updated model by using dotted lines (please refer to Respond Fig. 2, or new Fig. 6). We proposed the new model as follow: "In CccR-producing donor cells, CccR negatively autorepresses its own expression with a feedback mechanism. Under competition conditions, CccR is released into the extracellular milieu to relieve the autorepression. Released CccR enters target cells by engaging the TonB-dependent outer membrane transporter FhuA in a bacteriocin-like manner. In the presence of many prey cells (Left), secreted CccR is preferentially internalized by prey cells to AMPylate the cell division protein FtsZ, leading to cell filamentation and growth arrest. Following the decrease in prey cells (Right), secreted CccR is internalized by sister cells to repress cccR expression by acting as a transcriptional regulator, informing the population that its production is unnecessary. Under this condition, CccR may also act as a global regulator that regulates expression of genes involved in iron acquisition, motility, and energy production to coordinate bacterial behaviors and increase bacterial fitness. Given that toxic CccR will inhibit the growth of prey cells, the transcriptional regulator activity of CccR in prey cells can be ignored".
Overall, I would like to re-iterate that this is a very nice study and with some changes it could made a big contribution to the T6SS field. Thank you for the opportunity to review this very nice paper! Response: Thank you very much for your insightful and professional comments which greatly improved our manuscript! Reviewer #4 (Remarks to the Author):